FIGURE 2.

D₂-like receptors fail to recycle. Agonist-induced endocytosis (A–C) and recycling properties (D–F) of the D₂-like receptors were analyzed using flow cytometry. Cells were labeled with M1-conjugated Alexa Fluor® 647 antibody for 20 min and were either left untreated or pretreated with antagonist (ant) (10 μm raclopride, D₂ (A) or 10 μm spiperone D₃ (B), or D₄ (C)) or PKC inhibitor (BIM 1 μm) for 30 min. Cells were then stimulated with DA (D₂ and D₄) or PMA (D₃) for 45 min, washed in PBS/EDTA to remove antibody from any remaining cell surface receptors, and either fixed to monitor the degree of endocytosis (A–C, DA, ant-DA, BIM-DA, PMA, ant-PMA, and BIM-PMA) or returned to the incubator in the presence of PBS/EDTA containing either antagonist (E and F, DA-ant and PMA-ant) or PKC inhibitor (DA-BIM and PMA-BIM) to monitor recycling. Data are represented as the mean of at least four independent experiments performed in duplicate analyzed using one-way ANOVA with Bonferroni t test. ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05 compared with untreated controls or ###, p ≤ 0.001; ##, p ≤ 0.01; or #, p ≤ 0.05 compared with DA/PMA-treated samples. ns, not significant.